Bottom Line:
As stem cells undergo differentiation, mitochondrial DNA (mtDNA) copy number is strictly regulated in order that specialized cells can generate appropriate levels of adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS) to undertake their specific functions.We show that human neural stem cells (hNSCs) increased their mtDNA content during differentiation in a process that was mediated by a synergistic relationship between the nuclear and mitochondrial genomes and results in increased respiratory capacity.However, prolonged depletion resulted in impaired mtDNA replication, reduced proliferation and induced the expression of early developmental and pro-survival markers including POU class 5 homeobox 1 (OCT4) and sonic hedgehog (SHH).

ABSTRACTAs stem cells undergo differentiation, mitochondrial DNA (mtDNA) copy number is strictly regulated in order that specialized cells can generate appropriate levels of adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS) to undertake their specific functions. It is not understood whether tumor-initiating cells regulate their mtDNA in a similar manner or whether mtDNA is essential for tumorigenesis. We show that human neural stem cells (hNSCs) increased their mtDNA content during differentiation in a process that was mediated by a synergistic relationship between the nuclear and mitochondrial genomes and results in increased respiratory capacity. Differentiating multipotent glioblastoma cells failed to match the expansion in mtDNA copy number, patterns of gene expression and increased respiratory capacity observed in hNSCs. Partial depletion of glioblastoma cell mtDNA rescued mtDNA replication events and enhanced cell differentiation. However, prolonged depletion resulted in impaired mtDNA replication, reduced proliferation and induced the expression of early developmental and pro-survival markers including POU class 5 homeobox 1 (OCT4) and sonic hedgehog (SHH). The transfer of glioblastoma cells depleted to varying degrees of their mtDNA content into immunocompromised mice resulted in tumors requiring significantly longer to form compared with non-depleted cells. The number of tumors formed and the time to tumor formation was relative to the degree of mtDNA depletion. The tumors derived from mtDNA depleted glioblastoma cells recovered their mtDNA copy number as part of the tumor formation process. These outcomes demonstrate the importance of mtDNA to the initiation and maintenance of tumorigenesis in glioblastoma multiforme.

fig4: Recovery of mtDNA copy number and gene expression in depleted GBM cells. Mean (±S.E.M.) mtDNA copy number following 14 days of recovery for undifferentiated HSR-GBM1 cells depleted for up to 50 days (a). Gene expression relative to β-ACTIN for HSR-GBM1 cells following 14 days of recovery after depletion for up to 25 days. Fold change in expression of NESTIN (b), MUSASHI1 (c), CD133 (d) and GFAP (e) is relative to non-depleted GBM cells. Bars represent mean values±S.E.M. *P<0.05, **P<0.01 and ***P<0.001

Mentions:
To determine whether replenishment of mtDNA copy number influenced gene expression of undifferentiated HSR-GBM1 cells, we analyzed cells depleted of mtDNA for 7, 14, 21, 25 and 50 days followed by recovery for 14 days (Figure 4a). HSR-GBM1 cells recovering after 7 days of depletion re-established copy number to levels 1.12 higher than non-depleted cells (P<0.05), demonstrating that mtDNA depletion is reversible. However, cells recovering from 14, 21 and 25 days failed to fully replenish copy number with levels 1.40-, 6.60- and 23.30-fold lower than for non-depleted cells, respectively (P<0.001). Cells depleted for 50 days failed to replenish mtDNA. Consequently, depletion as far as 7 to 14 days enables cells to fully replenish mtDNA. However, recovery of cells in conditioned media from non-depleted cells enabled cells depleted for 14 and 21 days to replenish copy number to levels significantly above or just below non-depleted cells, respectively (Supplementary Figure S3A).

fig4: Recovery of mtDNA copy number and gene expression in depleted GBM cells. Mean (±S.E.M.) mtDNA copy number following 14 days of recovery for undifferentiated HSR-GBM1 cells depleted for up to 50 days (a). Gene expression relative to β-ACTIN for HSR-GBM1 cells following 14 days of recovery after depletion for up to 25 days. Fold change in expression of NESTIN (b), MUSASHI1 (c), CD133 (d) and GFAP (e) is relative to non-depleted GBM cells. Bars represent mean values±S.E.M. *P<0.05, **P<0.01 and ***P<0.001

Mentions:
To determine whether replenishment of mtDNA copy number influenced gene expression of undifferentiated HSR-GBM1 cells, we analyzed cells depleted of mtDNA for 7, 14, 21, 25 and 50 days followed by recovery for 14 days (Figure 4a). HSR-GBM1 cells recovering after 7 days of depletion re-established copy number to levels 1.12 higher than non-depleted cells (P<0.05), demonstrating that mtDNA depletion is reversible. However, cells recovering from 14, 21 and 25 days failed to fully replenish copy number with levels 1.40-, 6.60- and 23.30-fold lower than for non-depleted cells, respectively (P<0.001). Cells depleted for 50 days failed to replenish mtDNA. Consequently, depletion as far as 7 to 14 days enables cells to fully replenish mtDNA. However, recovery of cells in conditioned media from non-depleted cells enabled cells depleted for 14 and 21 days to replenish copy number to levels significantly above or just below non-depleted cells, respectively (Supplementary Figure S3A).

Bottom Line:
As stem cells undergo differentiation, mitochondrial DNA (mtDNA) copy number is strictly regulated in order that specialized cells can generate appropriate levels of adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS) to undertake their specific functions.We show that human neural stem cells (hNSCs) increased their mtDNA content during differentiation in a process that was mediated by a synergistic relationship between the nuclear and mitochondrial genomes and results in increased respiratory capacity.However, prolonged depletion resulted in impaired mtDNA replication, reduced proliferation and induced the expression of early developmental and pro-survival markers including POU class 5 homeobox 1 (OCT4) and sonic hedgehog (SHH).

ABSTRACTAs stem cells undergo differentiation, mitochondrial DNA (mtDNA) copy number is strictly regulated in order that specialized cells can generate appropriate levels of adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS) to undertake their specific functions. It is not understood whether tumor-initiating cells regulate their mtDNA in a similar manner or whether mtDNA is essential for tumorigenesis. We show that human neural stem cells (hNSCs) increased their mtDNA content during differentiation in a process that was mediated by a synergistic relationship between the nuclear and mitochondrial genomes and results in increased respiratory capacity. Differentiating multipotent glioblastoma cells failed to match the expansion in mtDNA copy number, patterns of gene expression and increased respiratory capacity observed in hNSCs. Partial depletion of glioblastoma cell mtDNA rescued mtDNA replication events and enhanced cell differentiation. However, prolonged depletion resulted in impaired mtDNA replication, reduced proliferation and induced the expression of early developmental and pro-survival markers including POU class 5 homeobox 1 (OCT4) and sonic hedgehog (SHH). The transfer of glioblastoma cells depleted to varying degrees of their mtDNA content into immunocompromised mice resulted in tumors requiring significantly longer to form compared with non-depleted cells. The number of tumors formed and the time to tumor formation was relative to the degree of mtDNA depletion. The tumors derived from mtDNA depleted glioblastoma cells recovered their mtDNA copy number as part of the tumor formation process. These outcomes demonstrate the importance of mtDNA to the initiation and maintenance of tumorigenesis in glioblastoma multiforme.